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Architecture and dynamics of a desmosome–endoplasmic reticulum complex

Nature Cell Biology volume 25, pages 823–835 (2023)Cite this article

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An Author Correction to this article was published on 12 February 2024

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Abstract

The endoplasmic reticulum (ER) forms a dynamic network that contacts other cellular membranes to regulate stress responses, calcium signalling and lipid transfer. Here, using high-resolution volume electron microscopy, we find that the ER forms a previously unknown association with keratin intermediate filaments and desmosomal cell–cell junctions. Peripheral ER assembles into mirror image-like arrangements at desmosomes and exhibits nanometre proximity to keratin filaments and the desmosome cytoplasmic plaque. ER tubules exhibit stable associations with desmosomes, and perturbation of desmosomes or keratin filaments alters ER organization, mobility and expression of ER stress transcripts. These findings indicate that desmosomes and the keratin cytoskeleton regulate the distribution, function and dynamics of the ER network. Overall, this study reveals a previously unknown subcellular architecture defined by the structural integration of ER tubules with an epithelial intercellular junction.

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Fig. 1: FIB-SEM reveals ER–desmosome associations.
Fig. 2: FIB-SEM reveals ER–keratin filament associations in A431 cells.
Fig. 3: Analysis of FIB-SEM data reveals a symmetrical organization of ER and keratin filaments proximal to desmosomes.
Fig. 4: Desmosomes anchor ER tubules and stabilize ER membrane.
Fig. 5: ER remains anchored to desmosomal junctions but retracts from cell-free edges when microtubules are depolymerized.
Fig. 6: ER tubules associate with desmosomes and keratin filaments during assembly.
Fig. 7: Desmosomes and keratin regulate peripheral ER organization and ER membrane stability.
Fig. 8: Keratin aggregation perturbs ER membrane morphology and alterations in keratin or desmosome function cause ER stress.

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Data availability

All macros and plasmid maps are available at https://doi.org/10.5281/zenodo.6800360 (ref. 39). FIB-SEM datasets are hosted on https://www.openorganelle.org/datasets/aic_desmosome-2 and https://www.openorganelle.org/datasets/aic_desmosome-3. Other imaging source data, including spinning disk confocal microscopy data, are hosted on ScholarSphere (https://doi.org/10.26207/53vq-g344). Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Code availability

All code and macros are available at https://doi.org/10.5281/zenodo.6800360.

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Acknowledgements

The authors are grateful to D. Lerit (Emory University), K. Green and L. Godsel (Northwestern University), T. Magin (University of Leipzig), M. Amagai (Keio University, Tokyo) and A. Nelson and R. Hobbs (Penn State College of Medicine) for instrument use, reagents and advice. We thank N. Sheaffer and J. Bednarczyk from Penn State College of Medicine’s Flow Cytometry Core for assistance with cell sorting. This research project was supported in part by the Emory University Integrated Cellular Imaging Core. This work was supported by: National Institutes of Health grant R01AR048266 (A.P.K.); Natural Sciences and Engineering Research Council of Canada Discovery Grant RGPIN-2018-14 03727 (A.W.V.). Cryo-SIM and FIB-SEM imaging were done in collaboration with the Advanced Imaging Center at Janelia Research Campus, a facility jointly supported by the Gordon and Betty Moore Foundation and the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Departments of Dermatology and Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA, USA

    Navaneetha Krishnan Bharathan, William Giang, Coryn L. Hoffman, Sara N. Stahley & Andrew P. Kowalczyk

  2. Advanced Imaging Center, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

    Jesse S. Aaron, Satya Khuon, Teng-Leong Chew, Stephan Preibisch, Eric T. Trautman, Larissa Heinrich, John Bogovic, Davis Bennett, David Ackerman, Woohyun Park, Alyson Petruncio, Aubrey V. Weigel & Stephan Saalfeld

  3. Life Sciences Institute and the Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada

    A. Wayne Vogl

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COSEM Project Team

  • Stephan Preibisch
  • , Eric T. Trautman
  • , Larissa Heinrich
  • , John Bogovic
  • , Davis Bennett
  • , David Ackerman
  • , Woohyun Park
  • , Alyson Petruncio
  • , Stephan Saalfeld
  •  & Aubrey V. Weigel

Contributions

N.K.B., S.N.S. and A.P.K. were involved with project conception. N.K.B. and A.P.K. wrote the manuscript with input from all co-authors. N.K.B. and W.G. were involved with experimental design, image acquisition and analysis, writing the methods section and making figures and movies. N.K.B. and C.L.H. performed qRT–PCR analysis. T.-L.C. supervised Cryo-SIM and FIB-SEM workflow. J.S.A. was involved with cryogenic light microscopy image acquisition/processing and FIB-SEM data acquisition. S.K. was involved with Cryo-SIM/FIB-SEM sample preparation including cell culture/labelling, high-pressure freezing and sample trimming. COSEM Project Team, S.S. and A.V.W. supervised ER segmentation in FIB-SEM datasets. S.P., E.T.T. and D.B. were involved with FIB-SEM data pre-processing. D.B. and E.T.T. organized FIB-SEM data and data attributes. W.P. and A.P. provided manual annotations, evaluations and proofreading. D.B. built the data management infrastructure. L.H. and S.S. developed machine learning algorithms; L.H. performed network training and predictions. D.A. and S.S. developed refinement and analysis algorithms; D.A. analysed data. J.B. and S.S. developed automated CLEM registration algorithms; J.B. performed automated CLEM registration. A.W.V. was involved with transmission EM acquisition in Extended Data Fig. 2. A.P.K. and S.N.S. were involved with funding acquisition. A.P.K. supervised the project.

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Correspondence to Andrew P. Kowalczyk.

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Extended data

Extended Data Fig. 1 ER tubules are proximal to desmosomal junctions in various epithelial cell culture models.

(a–c) Representative light microscopy images of a pair of A431 cells (A), HaCaT immortalized keratinocytes (B), and primary normal human epidermal keratinocytes (C) expressing mCherry-VAPB (magenta, ER marker) showing ER tubule and desmosomal junction (orange) proximity (red or white arrowheads). Desmosomes are labelled with DP-EGFP in A and B, or with an Alexa Fluor 488 conjugated anti-DSG3 mAb (AK23) in C. Images are representative of n = 3 independent experiments (A, B) and 2 independent experiments (C). Scale bar = 2μm.

Extended Data Fig. 2 ER tubules are proximal to the desmosomal plaque in epithelial cell culture models and various tissues.

(a-h) Transmission electron micrographs of A431 epithelial cells (A-D), rat skin (E, F), and rat enterocytes (G, H) show ER tubules (red arrows) proximal to the electron-dense desmosomal plaque. Images are representative of n = 1 replicate for each cell or tissue type. Scale bar = 200 nm (A-F), 100 nm (G, H).

Extended Data Fig. 3 FIB-SEM reveals ER tubules in physical contact with the desmosome outer dense plaque.

(a–c) Orthoslices in XY (A), XZ (B), and YZ (C) showing ER tubules making physical contact with desmosome outer dense plaque. (d–f) Same orthoslices as in A-C with ER, keratin, and desmosome segmentations. Yellow arrows point to physical contact between ER tubules and the desmosome outer dense plaque in all 3 views. Images are representative of n = 1 cell-cell contact. Scale bar = 250 nm.

Extended Data Fig. 4 Ribosomes are less prominent on desmosome-adjacent ER than planar ER.

(a, b) Rotated views showing that ER structures (magenta) have fewer bound ribosomes (white) at desmosome (yellow)-adjacent regions (white arrows) than at regions further away from the desmosome (blue arrows) in both top and bottom cells. Images are representative of n = 1 cell-cell contact. Scale bar = 1μm. (c–e) Three-dimensional reconstructions of segmentations in 4 × 4 × 4nm3 FIB-SEM data reveal keratin filaments (teal) contacting and penetrating planar ER sheets (magenta). Microtubules (orange) are also present in the vicinity of planar ER, and ribosomes (white) decorate the surface of ER sheets. (f–h) Rotated views of the same region in C-E. Images are representative of n = 1 cell. Scale bar = 500 nm (C-H).

Extended Data Fig. 5 The ER makes more contacts with keratin filaments compared to microtubules proximal to the desmosome.

(a–b) FIB-SEM segmentations showing keratin filaments (teal), desmosomes (yellow), microtubules (orange), and ER (magenta) (A). Panel (B) displays ER-keratin (teal) and ER-microtubule (orange) contacts at a distance threshold of 16 nm. Scale bar = 1μm. (c–f) Close-up of keratin filaments, microtubules, and ER adjacent to a desmosome (C-E) revealing larger and more numerous ER-keratin contacts (blue arrowheads) compared to ER-microtubule contacts (orange arrowheads) (F). Images in A-F are representative of n = 1 cell-cell contact. Scale bar = 500 nm. (g) Violin plots of ER-keratin (blue) and ER-microtubule (orange) contacts at x nm away from the closest desmosome. Individual contacts are represented by black vertical lines. Setting various contact site distance thresholds between 0 nm to 16 nm (y-axis) demonstrates that ER-keratin contacts preferentially occur proximal to desmosomes. Source numerical data are available in source data.

Source data

Extended Data Fig. 6 ER tubules closely associate with desmosomes in live cells.

(a–e) Snapshots of a live-cell time-course of Desmoplakin-EGFP (DP) (top row, orange in bottom row) and mApple-VAPB (middle row, magenta in bottom row) in A431 cells at the cell-cell contact over 2 minutes (Images are representative of n = 3 independent experiments). Gamma correction was applied to grayscale images. Gamma (inverted) = 1.5 (VAPB). Scale bar = 2μm. (f–j) ilastik-segmentations of DP (top row) and VAPB (middle row) channels in A-E. Bottom row indicates overlapping pixels (white) between DP and VAPB channels. Overlap indicates that tips of ER tubules colocalize with DP puncta. Scale bar = 2μm.

Extended Data Fig. 7 ER retracts from cell-free edges only in cells where microtubules are depolymerized.

(a, b) Representative images of A431 cells treated with either DMSO or 30μM nocodazole reveal that ER (VAPB, magenta) tubules are converted to sheets with nocodazole treatment. The ER remains tethered to desmosomes (DP, orange) at cell-cell contacts under both treatment conditions. Images are representative of at least n = 2 independent experiments. Scale bar = 2μm. (c, d) Representative images of A431 cells treated with either DMSO or 30μM nocodazole reveal that ER (VAPB, magenta) remains close to the periphery of a cell-free edge in DMSO-treated cells (C) but retracts in nocodazole-treated cells (D). Tubulin Tracker Deep Red labelling reveals a lack of microtubules in nocodazole-treated cells but not in DMSO-treated cells. Cell-free edge is depicted by a dashed red or white line. Images are representative of at least n = 2 independent experiments. Scale bar = 2μm. Gamma correction was applied to some grayscale images. Gamma (inverted) = 1.75 (D, DP channel), 0.75 (A, Tubulin Tracker channel), 1.5 (C, Tubulin Tracker channel).

Extended Data Fig. 8 ER tubules associate with desmosomes during fusion events.

(a–f) Snapshots of a live-cell time-course of desmoplakin (top row, orange in bottom row) and VAPB (middle row, magenta in bottom row) in A431 cells during the fusion of DP puncta at the cell-cell contact (Images are representative of n = 4 independent experiments). Three DP puncta (A) fuse to form two puncta (B). Eventually, these puncta fuse to form one DP puncta (F). Gamma correction was applied to grayscale images. Gamma = 0.7 (DP/ VAPB merge), Gamma (inverted) = 1.5 (VAPB). Scale bar = 1μm. (g–l) ilastik-segmentations of DP (top row) and VAPB (middle row) channels in A-F. Bottom row indicates overlapping pixels (white) between DP and VAPB channels. Overlap indicates that tips of ER tubules colocalize with DP puncta during fusion. Scale bar = 1μm.

Extended Data Fig. 9 ER tubules remain at the cell-cell border even when desmoplakin translocation to the border is inhibited.

(a–e) Representative time-lapse of A431 cells expressing DP-EGFP (orange) and mApple-VAPB (magenta) pre-treated with DMSO for 30 minutes, followed by a switch to high calcium media. ER tubules extend toward the cell periphery, sometimes forming paired structures across adjacent cells (red arrows, B). Desmosomes form at the exact position of ER paired structures (blue arrows in E). Images are representative of n = 2 independent experiments. Scale bar = 2μm. (f–j) Representative time-lapse of A431 cells expressing DP-EGFP (orange) and mApple-VAPB (magenta) pre-treated with 1μM thapsigargin (TG) for 30 minutes, followed by a switch to high calcium media. ER tubules extend toward the cell periphery, sometimes forming paired structures across adjacent cells (red arrows, F, G). ER tubules eventually remain at the periphery in adjacent cells, but no desmosomes form (J). Timestamps indicate time after DMSO/ thapsigargin wash-out and switch to high calcium media. Images are representative of n = 3 independent experiments. Scale bar = 2μm.

Extended Data Fig. 10 Keratin filaments and aggregates are stably tethered to ER membranes.

(a) Snapshot of an A431 cell expressing mNeonGreen-KRT14WT (blue) and mApple-VAPB (magenta) showing ER tubules along keratin filaments (Images are representative of n = 3 independent experiments). Solid yellow line indicates position of kymograph in B-D. Scale bar = 1μm. (b-d) Kymograph of yellow line in A revealing stable ER-keratin interaction over a 2-minute time course. Scale bar = 500 nm. (e) Snapshot of an A431 cell expressing mNeonGreen-KRT14R125C (blue) and mApple-VAPB (magenta) showing ER sheets surrounding KRT14R125C aggregates (Images are representative of n = 3 independent experiments). Solid yellow line indicates position of kymograph in F-H. Scale bar = 1μm. (f–h) Kymograph of yellow line in E revealing stable ER-keratin interactions over a 2-minute time course. Scale bar = 500 nm. (i) Graph depicting the fraction of KRT14R125C aggregates colocalizing with ER at each timepoint during a 25 timepoint/2-minute time course. Data are represented as mean ± s.d.; n indicates total number of KRT14R125C aggregates analyzed from three independent experiments. Source numerical data are available in source data.

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Supplementary Figs. 1–4.

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Supplementary Video 1

FIB-SEM reveals a desmosome–ER complex. Slice-by-slice view of 4 × 4 × 4 nm3 FIB-SEM dataset ‘DSM-3’ at the cell–cell contact in A431 epithelial cells shows raw data (bottom) and overlaid segmentations of desmosome outer dense plaques (yellow), keratin filaments (teal), microtubules (orange) and endosomes (green) (top). ER tubules (magenta) are closely associated with desmosome plaques and keratin filaments. Scale bar, 1 μm. As an orientation aid, a 3D rendering of the same dataset is shown in the lower right corner along with an outline of the current slice position. In the second part of the video, a magnified 3D rendering of the same dataset shows the PM (white) with apical protrusions. ER tubules approach the desmosome plaque and form paired structures on either side of the desmosome.

Supplementary Video 2

FIB-SEM reveals ER–desmosome contact. Segmentations reveal ER tubules (magenta) in the space between the keratin filaments (teal) and the desmosome outer dense plaque (yellow). ER tubules are in contact with the desmosome.

Supplementary Video 3

FIB-SEM reveals ER–keratin association. Segmentations reveal that peripheral ER tubules (magenta) wrap around the keratin filaments (teal), making physical contact. Towards the cell interior, the ER is more planar/sheet-like and is penetrated by keratin filaments.

Supplementary Video 4

FIB-SEM reveals the organization of other organelles and cytoskeletal filaments at the cell–cell contact. Segmentations reveal that endosomes (green) are present near the cell–cell contact. Occasionally, ER tubules (magenta) wrap around endosomes. Microtubules (orange) also associate with the ER near the cell–cell contact.

Supplementary Video 5

ER tubules form stable mirror image-like arrangements at desmosomal junctions. Live-cell spinning disk confocal microscopy of A431 epithelial cells shows that ER tubules (magenta) are stably anchored to DP puncta (orange) over a 2 min duration forming a mirror image-like arrangement (yellow arrowheads) on either side of the cell–cell contact. Scale bar, 2 μm.

Supplementary Video 6

Microtubule depolymerization leads to retraction of ER from cell-free edges, but not from desmosomes at the cell–cell contact. Live-cell spinning disk confocal microscopy of A431 epithelial cells treated with nocodazole shows that the ER (magenta) is more sheet-like when microtubules (white) depolymerize. ER sheets still persist at DP puncta (orange) but retract from cell-free edges. Scale bar, 2 μm.

Supplementary Video 7

ER tubules associate with desmosomes during fusion events. Live-cell spinning disk confocal microscopy of A431 epithelial cells shows that ER tubules (magenta) persist at DP puncta (orange) as puncta undergo fusion (yellow box in left panel, red box in middle and right panel). Scale bar, 4 μm.

Supplementary Video 8

ER tubules associate with desmosomes during cell–cell contact formation. Live-cell spinning disk confocal microscopy of A431 epithelial cells shows ER tubules (magenta) forming mirror image-like arrangements at sites of nascent DP puncta formation (orange) (yellow arrowhead in left panel, black arrowheads in middle and right panel). Scale bar, 2 μm.

Supplementary Video 9

ER tubules associate with keratin filaments during cell–cell contact formation. Live-cell spinning disk confocal microscopy of A431 epithelial cells shows ER tubules (magenta) and keratin filaments (blue) extending together as cells form contacts (at 2.5 min). Eventually, at 29.5 min, ER tubules and keratin filaments form mirror image-like structures at cell–cell contacts (yellow arrowhead in left panel, black arrowheads in middle and right panel). Scale bar, 2 μm.

Supplementary Video 10

Desmosomes regulate peripheral ER tubule organization. Live-cell spinning disk confocal microscopy of a pair of A431 WT cells (top row) or A431 DSG2-null cells (bottom row). In A431 WT cells, keratin filaments (blue) and ER tubules (magenta) extend radially towards the cell–cell contact (yellow arrowheads). In A431 DSG2-null cells, keratin filaments (blue) and ER tubules (magenta) are perpendicular to the cell–cell contact (red arrowheads). Scale bar, 4 μm.

Supplementary Video 11

Keratin filaments regulate peripheral ER morphology. Live-cell spinning disk confocal microscopy of a pair of A431 cells expressing KRT14WT (top row) or the EBS mutant KRT14R125C (bottom row). In KRT14WT cells, keratins form filaments (blue) and peripheral ER morphology is tubular in nature. In KRT14R125C cells, keratins form aggregates (blue) and peripheral ER morphology is planar/sheet-like in nature. Scale bar, 4 μm.

Supplementary Video 12

ER membranes stably associate with keratin filaments or aggregates. Live-cell spinning disk confocal microscopy of a mix of A431 cells expressing KRT14WT (top and left cells) or the EBS mutant KRT14R125C (bottom right cell). In KRT14WT cells, keratins form filaments (blue) and stably associate with peripheral ER tubules (yellow box). In KRT14R125C cells, keratins form aggregates (blue) and stably associate with planar ER membranes (red box). Scale bar, 4 μm.

Supplementary Table 1

This file contains Supplementary Tables 1–8, each in its own tab combined into one Excel workbook.

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Source Data Extended Data Fig. 5

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Bharathan, N.K., Giang, W., Hoffman, C.L. et al. Architecture and dynamics of a desmosome–endoplasmic reticulum complex. Nat Cell Biol 25, 823–835 (2023). https://doi.org/10.1038/s41556-023-01154-4

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